This invention was made in part during work partially supported by the U.S. Department of Energy under DOE Contract No.: DE-AC03-76SF00098. The government has certain rights in the invention.
The present invention relates to methods and compositions for the direct detection of membrane conformational changes through the detection of color changes in biopolymeric materials. In particular, the present invention allows for the direct colorimetric detection of membrane modifying reactions and analytes responsible for such modifications and for the screening of reaction inhibitors.
Measuring and identifying the activity of various enzymes and other molecules involved in membrane rearrangement (e.g., lipid cleavage, polymerization, lipid flipping, transmembrane signalling, vesicle formation, lipidation, glycosylation, ion channeling, molecular rearrangement, and phosphorylation, among others) is important for the development of methods and compositions for regulating membrane biology and associated processes (e.g., signal transduction). Such methods and compositions will find use in regulating and treating numerous conditions (e.g., cancer, diabetes, viral infection, and obesity to name a few) and physiological processes (e.g., memory, aging, and metabolism to name a few).
Interfacial catalysis provides one example of such membrane reorganization and illustrates the benefits and limitations of current technologies in characterizing and exploiting these membrane reorganizations. Interfacial catalysis on biomembranes covers a range of enzyme classes such as lipolytic enzymes, acyltransferases, protein kinases, and glycosidases, and plays a key role in extra- and intracellular processes. In particular, lipolytic enzymes are involved in important biochemical processes including fat digestion and signal transduction. Recent interest in one such enzyme, phospholipase A2 (PLA2) (See e.g., Kini, Venom Phospholipase A2 Enzymes, Wiley, Chichester [1997]; and Waite, The Phospholipases, Plenum Press, New York [1987]) is motivated by its role in the release of arachidonate and lysophospholipids from membranes. These compounds are the precursors for the biosynthesis of eicosanoids (e.g, prostaglandins, leukotrienes, and hydroperoxy fatty acids) that have been implicated in a range of inflammatory diseases such as asthma, ischaemia, and rheumatoid arthritis (See e.g., Bomalaski and Clark, Arthritis and Rheumatism 36, 190 [1993]; Ramirez and Jain, Proteins: Structure Function, and Genetics, 9, 229 [1991]; and Dennis and Wong, Phospholipase A2: Role and Function in Inflammation, Plenum, New York [1990]) and are likely involved in a host of other physiological processes ranging from vision (See e.g., Camras et al., Ophthamology 103, 1916 [1996]), platelet aggregation (See e.g., Wu, J. Formos. Med. Assoc. 95, 661 [1996]), adipocyte differentiation (See e.g., Casimir et al., Differentiation 60, 203 [1996]), and luteolysis (See e.g., Tsai and Wiltbank, Biol. Reprod. 57, 1016 [1997]). Accordingly, the identification of PLA2 inhibitors is an active area of current research that may lead to the development of novel therapeutics and new biochemical insights into the mechanisms of enzyme activity (Dennis, supra; Gelb et al., FASEB Journal 8, 916 [1994]; and Lin and Gelb, J. Am. Chem. Soc. 115, 3932 [1993]).
PLA2 catalyzes the hydrolysis of an acyl ester bond exclusively at the 2-acyl position in glycerophospholipids, yielding free fatty acid and lysophospholipid. Typical methods for measuring this activity include discontinuous radiochemical (Ehnholm and Kuusi, Meth. Enzymol, 129, 716 [1986]), fluorescent (Bayburt et al., Analytical Biochemistry, 232, 7 [1995]), and spectrophotometric techniques (Reynolds et al., Analytical Biochemistry 204, 190 [1992]). In these measurements, labeled acyl phospholipids are used as substrates, and enzyme activity is evaluated by the radioactivity, fluorescence, or absorbance of the cleaved fatty acids. Some procedures, and particularly radiolabel methods, may require that the cleaved fatty acids be extracted and isolated from the unreacted substrate by thin layer chromatography of HPLC. The extraction step and the need for synthetic labeled substrates are disadvantages when considering rapid analysis of enzyme activity, for example in high throughput assays that screen potential enzyme inhibitors. Furthermore, phospholipase catalysis is sensitive to the chemical structure of the phospholipid substrate (Grainger et al., Biochimica et Biophysica Acta 1022, 146 [1990]; and Wu and Cho, Analytical Biochemistry 221, 152 [1994]). Therefore the use of non-labeled naturally occurring substrates is highly desirable.
This need for non-labeled naturally occurring substrates applies not only to phospholipase A2 characterization, but also to other phospholipases (e.g., phospholipase C and phospholipase D), lipases in general (e.g., triacylglycerol lipases, lipoprotein lipases, and pancreatic lipases), other membrane modifing enzymes (e.g., lipolytic enzymes, acyltransferases, protein kinases, and glycosidase), and any other natural or artificial membrane modifying events. In particular, methods and compositions that provide simple detection of the modifying events and that allow high throughput screening of inhibitors are desired.
The present invention relates to methods and compositions for the direct detection of membrane conformational changes through the detection of color changes in biopolymeric materials. In particular, the present invention allows for the direct colorimetric detection of membrane modifying reactions and analytes responsible for such modifications and for the screening of reaction inhibitors.
The presently claimed invention provides methods for detecting a reaction, comprising: providing biopolymeric material comprising reaction substrate and a plurality of self-assembling monomers, and a reaction means; exposing the reaction means to the biopolymeric material; and detecting a color change in the biopolymeric material which indicates at least a partial occurrence of the reaction. In some embodiments, the method further comprises the step of quantifying the color change in the biopolymeric material.
In some embodiments, the reaction means comprises a lipid cleavage means. In particular embodiments, the cleavage means comprises a lipase. In specific embodiments, the lipase is selected from the group consisting of phospholipase A2, phospholipase C, and phospholipase D.
The presently claimed invention provides methods wherein the biopolymeric materials are selected from the group consisting of liposomes, films, tubules, helical assemblies, fiber-like assemblies, and solvated polymers. In some embodiments, the self assembling monomers of the biopolymeric materials comprise diacetylene monomers. In some embodiments, the self assembling monomers comprise diacetylene monomers selected from the group consisting of 5,7-docosadiynoic acid, 5,7-pentacosadiynoic acid, 10,12-pentacosadiynoic acid, and combinations thereof. In other embodiments, the self-assembling monomers are selected from the group consisting of acetylenes, alkenes, thiophenes, polythiophenes, siloxanes, poly-silanes, anilines, pyrroles, polyacetylenes, poly (para-phylenevinylene), poly (para-phylene), vinylpyridinium, and combinations thereof.
The presently claimed invention provides methods wherein the biopolymeric material further comprises one or more ligands. In some embodiments, the ligand is selected from the group consisting of proteins, antibodies, carbohydrates, nucleic acids, drugs, chromophores, antigens, chelating compounds, short peptides, pepstatin, Diels-Alder reagents, molecular recognition complexes, ionic groups, polymerizable groups, linker groups, electron donors, electron acceptor groups, hydrophobic groups, hydrophilic groups, receptor binding groups, trisaccharides, tetrasaccharides, ganglioside GM1, ganglioside GT1b, sialic acid, and combinations thereof. In certain embodiments, the ligands have affinity for the reaction means.
The presently claimed invention also provides methods wherein the biopolymeric material further comprises one or more dopants. In some embodiments, the dopant is selected from the group consisting of surfactants, polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol, zwitterionic detergents, decylglucoside, deoxycholate, diacetylene derivatives, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, steroids, cerebroside, lysophosphatidylcholine, D-erythroshingosine, sphingomyelin, dodecyl phosphocholine, N-biotinyl phosphatidylethanolamine and combinations thereof. In specific embodiments, the dopants comprise diacetylene derivatives selected from the group consisting of sialic acid-derived diacetylene, lactose-derived diacetylene, amino acid-derived diacetylene, and combinations thereof.
In some embodiments, the biopolymeric material further comprises a support, wherein the biopolymeric material is immobilized to the support. In particular embodiments, the support is selected from the group consisting of polystyrene, polyethylene, teflon, mica, sephadex, sepharose, polyacrynitriles, filters, glass, gold, silicon chips, and silica.
The presently claimed invention further provides methods for detecting the presence of an analyte, comprising providing biopolymeric material comprising analyte substrate and a plurality of self-assembling monomers; exposing a sample suspected of containing the analyte to the biopolymeric material; and detecting a color change in the biopolymeric material, which indicates the presence of the analyte. In some embodiments, the analyte comprises a lipid cleavage means. In particular embodiments, the cleavage means comprises a lipase. In specific embodiments, the lipase is selected from the group consisting of phospholipase A2, phospholipase C, and phospholipase D. In some embodiments, the biopolymeric material further comprises one or more ligands. In certain embodiments, the ligands have affinity for the analyte.
The presently claimed invention further provides methods for detecting inhibitors, comprising: providing biopolymeric material comprising reaction substrate and a plurality of self-assembling monomers, a reaction means, and a sample suspected of containing an inhibitor; combining the biopolymeric material and the sample suspected of containing an inhibitor; exposing the biopolymeric material and the sample suspected of containing an inhibitor to the reaction means; and detecting a color change in the biopolymeric material, thereby detecting the activity of the inhibitor. In some embodiments, the detecting a color change in the biopolymeric material comprises comparing the color change to one or more control samples. In some embodiments, the method further comprises the step of quantitating the color change in the biopolymeric material.
In some embodiments, the reaction means comprises a lipid cleavage means. In particular embodiments, the cleavage means comprises a lipase. In specific embodiments, the lipase is selected from the group consisting of phospholipase A2, phospholipase C, and phospholipase D.